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Elevated Expression of PGR5 and NDH-H in Bundle Sheath Chloroplasts in C4Flaveria Species

Elevated Expression of PGR5 and NDH-H in Bundle Sheath Chloroplasts in C4Flaveria Species Abstract Cyclic electron transport around PSI has been proposed to supply the additional ATP required for C4 photosynthesis. To investigate the nature of cyclic electron pathways involved in C4 photosynthesis, we analyzed tissue-specific expression of PGR5 (PROTON GRADIENT REGULATION 5), which is involved in the antimycin A-sensitive pathway, and NDH-H, a subunit of the plastidial NAD(P)H dehydrogenase complex, in four Flaveria species comprising NADP-malic enzyme (ME)-type C4, C3–C4 intermediate and C3 species. PGR5 was highly expressed in the C4 species and enriched in bundle sheath chloroplasts together with NDH-H, suggesting that electron transport of both PGR5-dependent and NDH-dependent cyclic pathways is promoted to drive C4 photosynthesis. C4 photosynthesis requires the coordinated functioning of two cell types, namely mesophyll and bundle sheath cells, with individual functions (Hatch 1987, Sage 1999). Although C4 photosynthesis suppress photorespiration by a CO2-concentrating mechanism, it increases the energetic cost of CO2 assimilation in comparison with C3 photosynthesis. Consequently, two extra ATP molecules are required for each CO2 molecule fixed to drive C4 photosynthesis (Kanai and Edwards 1999). The extra ATP needed for C4 photosynthesis was suggested to be produced by PSI cyclic electron transport activity, which contributes to generation of ΔpH across the thylakoid membrane (Kanai and Edwards 1999). Increased PSI cyclic electron transport compared with C3 plants has been reported in a number of C4 plants, including Sorghum bicolor and Zea mays (Herbert et al. 1990, Asada et al. 1993). Two cyclic pathways around PSI have been identified in C3 plants; the first pathway involves a plastidial NDH [NAD(P)H dehydrogenase] complex that is able to reduce plastoquinones from stromal NAD(P)H donors (Horvath et al. 2000) and the second pathway is an antimycin A-sensitive pathway involving PROTON GRADIENT REGULATION 5 (PGR5), which is localized in the chloroplast and considered to be a factor for major cyclic electron transport activity in C3 plants (Munekage et al. 2002, Munekage et al. 2004, Munekage et al. 2008). The contribution of NDH-dependent electron transport to C4 photosynthesis was suggested by its expression profile correlating with predicted ATP requirement in different cell types (Kubicki et al. 1996, Takabayashi et al. 2005). The contribution of PGR5-related electron transport to C4 photosynthesis is unclear. However, Ivanov et al. (2007) reported that the oxidation level of P700 in bundle sheath strands isolated from Z. mays was dependent on antimycin A. With the aim of investigating the nature of cyclic electron pathways involved in C4 photosynthesis, we analyzed expression of PGR5 and NDH-H, a subunit of the NDH complex, in the dicot genus Flaveria (Asteraceae), which contains closely related C3, C3–C4 intermediate and NADP-malic enzyme (ME)-type C4 species and is a widely used model system for studying the C4 photosynthesis evolutionary process (Sage 2004). The relative abundances of PGR5 and NDH-H were analyzed by immunoblotting in a C3-type species, F. pringlei, a C3–C4 intermediate species, F. anomala, and two NADP-ME C4-type species, F. trinervia and F. bidentis (Fig. 1). The abundance of major electron transport complexes was analyzed using specific antibodies against PsbO for PSII, the Rieske protein for the cytochrome b6f complex, and PsaC for PSI (Fig. 1). The C3F. pringlei and the C3–C4 intermediate F. anomala showed a similar relative abundance of PGR5, NDH-H and subunits of the major electron transport chain. In contrast, relative amounts of PGR5 and NDH-H in both C4 species were four and eight times higher than in C3–C4 intermediate and C3 species, respectively. While PsbO was less abundant in C4 species than in C3 and C3–C4 intermediate species, PsaC was slightly more abundant in C4 species. The abundance of the Rieske protein was similar among all the species compared. Fig. 1 View largeDownload slide Immunoblot analysis of PGR5, NDH-H, PsbO, Rieske protein and PsaC in C4F. trinervia, C4F. bidentis, C3–C4F. anomala and C3F. pringlei. Total membrane proteins were extracted from leaves of each Flaveria species. Lanes were loaded with 20 μg of proteins for detection of PGR5 and NDH-H, 5 μg of protein for detection of PsbO, 10 μg of proteins for detection of Rieske protein and PsaC, and a dilution series of F. bidentis as indicated. Fig. 1 View largeDownload slide Immunoblot analysis of PGR5, NDH-H, PsbO, Rieske protein and PsaC in C4F. trinervia, C4F. bidentis, C3–C4F. anomala and C3F. pringlei. Total membrane proteins were extracted from leaves of each Flaveria species. Lanes were loaded with 20 μg of proteins for detection of PGR5 and NDH-H, 5 μg of protein for detection of PsbO, 10 μg of proteins for detection of Rieske protein and PsaC, and a dilution series of F. bidentis as indicated. To compare the amino acid sequence of PGR5 between C3 and C4 species, the full-length PGR5 gene was cloned from cDNA libraries of F. trinervia, F. bidentis and F. pringlei. Alignment of the deduced protein sequences using the ClustalW algorithm (Supplementary Fig. S1) shows that the sequence is highly conserved among C3 and C4Flaveria. The program TARGETP (www.cbs.dtu.dk/services/TargetP) predicted that the N-terminal sequence of these PGR5 homologs contains a chloroplast-targeted transit peptide. The C-terminal sequence used to raise the PGR5 antibody (from Ala102 to Leu121) was 100% identical among the Flaveria species, indicating that the variation in immunodetection signal intensity reflects actual differences in PGR5 protein levels. To investigate the localization of PGR5 and the NDH complex in C3 and C4Flaveria, in situ immunolabeling was performed using both anti-PGR5 and anti-NDH-H antibodies (Fig. 2). Transverse sections of the leaf lamina of each Flaveria species were stained with toluidine blue. Numerous chloroplasts were colored blue in the mesophyll and bundle sheath cells (Fig. 2A–C). Transverse sections, prepared from the same leaf samples, were labeled with either the pre-immune serum or the immune serum and subsequently labeled with secondary antibodies conjugated to fluorescein isothiocyanate (FITC). Overlaid images of the FITC fluorescence in green and autofluorescence in red were visualized by confocal microscopy. The background labeling with pre-immune serum was very low in all cases (Fig. 2G–H, M, N) compared with the control section (labeling without primary antibody, Fig. 2D–F). There was very little immunolabeling for PGR5 in the C3F. pringlei (Fig. 2J). Although FITC fluorescence was observed in the vascular bundle, which did not contain chloroplasts (Fig. 2J), similar FITC fluorescence patterns were observed in leaf transverse sections of the Arabidopsis pgr5 mutant lacking PGR5 protein (data not shown), indicating that the FITC fluorescence observed in the vascular bundle was not caused by the PGR5 protein. In F. bidentis, more intense immunolabeling for PGR5 was observed in bundle sheath cells compared with mesophyll cells (Fig. 2K). The strong FITC fluorescence superimposed onto chloroplasts resulted in yellow fluorescence and indicated specific immunolabeling for PGR5. A similar result was obtained for F. trinervia (Fig. 2L). These results showed that PGR5 was enriched in bundle sheath chloroplasts of C4Flaveria. Immunolabeling for NDH-H showed exclusive localization of NDH-H in bundle sheath chloroplasts of the C4F. bidentis but only very faint staining of NDH-H in mesophyll cells of the C4F. bidentis and C3F. pringlei (Fig. 2O, P). Fig. 2 View largeDownload slide In situ immunolocalization of PGR5 and NDH-H in leaf tissue of C3F. pringlei (A, D, G, J, M, O), C4F. bidentis (B, E, H, K, N, P) and C4F. trinervia (C, F, I, L). Transverse sections of the lamina for anatomical observation were stained with toluidine blue (A–C). Localization of PGR5 and NDH-H was visualized by the green fluorescence of the FITC-labeled antibody. Leaf sections were stained with primary anti-PGR5 serum (J–L), anti-NDH-H serum (O, P) or without primary antibody (D–F). Pre-immunization sera for PGR5 (G–I) and for NDH-H (M, N) were used to analyze background labeling. Leaf sections were subsequently stained with secondary antibody (anti-rabbit-IgG–FITC conjugate). Overlaid images of green FITC fluorescence and red auto fluorescence were visualized by confocal microscopy. Scale bars = 100 μm. Fig. 2 View largeDownload slide In situ immunolocalization of PGR5 and NDH-H in leaf tissue of C3F. pringlei (A, D, G, J, M, O), C4F. bidentis (B, E, H, K, N, P) and C4F. trinervia (C, F, I, L). Transverse sections of the lamina for anatomical observation were stained with toluidine blue (A–C). Localization of PGR5 and NDH-H was visualized by the green fluorescence of the FITC-labeled antibody. Leaf sections were stained with primary anti-PGR5 serum (J–L), anti-NDH-H serum (O, P) or without primary antibody (D–F). Pre-immunization sera for PGR5 (G–I) and for NDH-H (M, N) were used to analyze background labeling. Leaf sections were subsequently stained with secondary antibody (anti-rabbit-IgG–FITC conjugate). Overlaid images of green FITC fluorescence and red auto fluorescence were visualized by confocal microscopy. Scale bars = 100 μm. In NADP-ME-type C4 photosynthesis, 3.3 ATP/2.1 NADPH molecules are estimated to be required per CO2 molecule fixed in mesophyll cells, which is a similar ratio to that in C3 photosynthesis, whereas only 2.3 ATP per CO2 fixed is considered to be required in bundle sheath cells since NADPH is supplied by decarboxylation of C4 acid (Kanai and Edward 1999). Increased expression of PGR5 and NDH-H in bundle sheath cells of NADP-ME-type C4Flaveria suggested promotion of both PGR5-dependent and NDH-dependent cyclic activities to fulfill the ATP requirement of C4 photosynthesis. In a previous study, expression profiles of NDH-H were well correlated with the predicted ATP requirement in C4 cell types, in contrast to PGR5. This was observed in both NAD-ME-type C4 plants and NADP-type C4 plants (Takabayashi et al. 2005). From these results, it was suggested that the NDH complex mainly energizes C4 photosynthesis. However, if the expression of PGR5 was normalized to the cytochrome f level, whose expression is comparable between mesophyll and bundle sheath cells (Kubicki et al., 1996, Majeran et al., 2008), the PGR5 expression could be correlated with the predicted ATP requirement in C4 cell types in those plants, with the exception of the case of NAD-ME-type Portulaca oleracea (Takabayashi et al. 2005). Here, we showed increased expression of PGR5 from the C3, over the C3–C4 intermediates to the C4 species of the genus Flaveria (Fig. 1B) and used an immunolabeling technique which showed enrichment of PGR5 protein in bundle sheath cells (Fig. 2). This result suggests that a PGR5-dependent pathway contributes to ATP production, which drives C4 photosynthesis. However, the abundances of PGR5, NDH-H and major electron transport complexes were similar between the C3–C4 intermediate F. anomala and C3F. pringlei. The proportion of PSII/PSI is similar between C3–C4 intermediate species and C3 species of Flaveria (Pfündel and Pfeffer 1997). Together, these findings suggest that the composition of the electron transport chain has remained unchanged during the evolution of C3 to C3–C4 intermediate species in Flaveria. In C4Flaveria, two morphologically distinct chloroplast types were observed, as in S. bicolor and Z. mays (Laetsch 1971, Hofer et al. 1992). While mesophyll chloroplasts contain numerous grana thylakoid membranes, bundle sheath chloroplasts contain grana-free thylakoid membranes (Laetsch 1971). Enrichment of PGR5 on bundle sheath chloroplasts suggests that PGR5 is probably localized on non-appressed thylakoid membranes. To test this hypothesis, thylakoid membranes isolated from C3F. pringlei or C4F. bidentis leaves were fractionated into grana and stroma lamellae by digitonin treatment (Cuello and Quiles 2004). SDS–PAGE revealed protein patterns typical for grana and stroma lamellae in C3F. pringlei; light- harvesting complex II (LHCII) was enriched in the grana thylakoid fraction, and α and β subunits of ATP synthase were enriched in the stroma lamellae fraction (Fig. 3A). A similar protein pattern was observed in the C4 species, with the exception of LHCIIs, which were also detected in the stroma lamellae fraction (Fig. 3A). Immunoblotting against Lhcb1 and the ε subunit of ATP synthase (AtpE) showed that Lhcb1 and AtpE were mainly localized in the grana thylakoid fraction and stroma lamellae fractions, respectively, in both F. pringlei and F. bidentis, but a small amount of Lhcb1 was also detected in stroma lamellae in F. bidentis (Fig. 3B). A higher amount of Lhcb1 was detected in the stroma lamellae fraction of F. bidentis than that of F. pringlei, which is probably due to localization of LHCIIs in agranal thylakoid membranes of bundle sheath chloroplasts in C4Flaveria (Hofer et al. 1992). Immunoblotting against PGR5 showed that PGR5 was enriched in the stroma lamellae fraction in both F. pringlei and F. bidentis (Fig. 3B). Identical results were obtained with tobacco (data not shown). From these results, we concluded that PGR5 is localized in stroma lamellae. Fig. 3 View largeDownload slide Distribution of PGR5 in grana and stroma thylakoids in C3F. pringlei and C4F. bidentis. (A) Coomassie blue staining of proteins extracted in the total thylakoids fraction (T), grana fraction (G) and stroma lamellae fraction (S). (B) Immunoblotting of PGR5, Lhcb1 and AtpE. The lanes were loaded with 10 μg of protein for Coomassie blue staining and immunoblotting of PGR5, and 0.5 μg of protein for immunoblotting of Lhcb1 and AtpE. Fig. 3 View largeDownload slide Distribution of PGR5 in grana and stroma thylakoids in C3F. pringlei and C4F. bidentis. (A) Coomassie blue staining of proteins extracted in the total thylakoids fraction (T), grana fraction (G) and stroma lamellae fraction (S). (B) Immunoblotting of PGR5, Lhcb1 and AtpE. The lanes were loaded with 10 μg of protein for Coomassie blue staining and immunoblotting of PGR5, and 0.5 μg of protein for immunoblotting of Lhcb1 and AtpE. Localization of PGR5 in stroma lamellae indicates that PGR5-related cyclic electron transport takes place in non-appressed thylakoid membranes. It also explains why NADP-ME-type C4 species developed agranal chloroplasts in bundle sheath cells to promote PSI cyclic electron transport. Interestingly, while expression of the PSI subunit was slightly higher in C4 species than in C3 species, expression of PGR5 was four times higher in C4 species than in C3 species (Fig. 1). We propose that the increased expression of PGR5 and the NDH complex directly promotes cyclic electron transport activity to drive ATP production for C4 photosynthesis in C4Flaveria species. Materials and Methods Plants of F. trinervia, F. bidentis, F. anomala and F. pringlei were grown in pots on soil for 6–8 weeks in a growth chamber (200–300 μmol photons m−2 s−1, 16 h light/8 h dark, 25–28°C). A cDNA library was prepared from leaves of F. bidentis. Total RNA was extracted from leaves using the RNeasy Maxi Kit (Qiagen, Courteboeuf, France) and poly(A) mRNA was isolated with the PolyATtract® mRNA Isolation System IV (Promega, Madison, WI, USA). cDNA synthesis and rapid amplification of cDNA ends (RACE) (3′ and 5′) PCR was performed using the Marathon™ cDNA Amplification Kit (Clontec, Ozyme, France) according to the manufacturer’s instructions. Primers for RACE PCR are listed in Supplementary Table S1. Leaf samples were blended in liquid N2 and the powder was suspended in 50 mM Tris–HCl buffer (pH 8.0) containing 50 mM β-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride. The membrane fraction was sedimented by centrifugation at 27,000 × g for 15 min and the pellet was resuspended in 50 mM Tris–HCl buffer (pH 8.0) containing 1% SDS. Stromal and granal thylakoid membrane separation was performed essentially as described by Rumeau et al. (2005). Leaves were homogenized at high speed using a Polytron homogenizer to grind mesophyll and bundle sheath cells. Isolated thylakoids were resuspended in 100 mM Tricine/NaOH buffer (pH 7.8) containing 10 mM NaCl and 10 mM MgCl2 to a chlorophyll concentration of 1.5 mg ml−1 and incubated for 3 min with digitonin (Sigma-Aldrich) added to give a final concentration of 8.25 mg ml−1. The samples were centrifuged at 10,000 × g for 30 min to separate grana membranes. After centrifugation at 40,000 × g for 30 min to remove contaminating grana vesicles, the supernatant (stroma lamellae) was sedimented at 150,000 × g for 1 h. Denaturing SDS–PAGE was performed using 15% (w/v) polyacrylamide gels. Proteins were electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with the following antibodies. The anti-NDH-H antibody raised from recombinant proteins and the anti-PGR5 antibody raised from a synthetic peptide antigen are described in Horvath et al. (2000) and Munekage et al. (2002), respectively. The anti-PsaC antibody and anti-Lhcb1 antibody were purchased from Agrisera (Vannas, Sweden). Antibodies against Rieske were raised from recombinant proteins (Sanda et al. unpublished data). Immunocomplexes were detected using the ECL Plus Western Blot Detection Reagent (GE Healthcare). Small leaf tissue fragments were first fixed in 3.7% formaldehyde, 5% acetic acid and 50% ethanol, dehydrated with ethanol, impregnated with xylene and then embedded in paraffin. Embedded tissue was sliced into 8 μm sections and mounted onto glass slides, deparaffinized and stained with toluidine blue for anatomical observation. For immunolabeling, deparaffinized sections were incubated in blocking solution [Tris-buffered saline with Tween (TBST) with 3% dry milk] then incubated with or without primary antibody (1 : 300 diluted in blocking solution). After washing in TBST, the sections were incubated with the secondary antibody (anti-rabbit-IgG–FITC conjugate, Sigma-Aldrich, 1 : 500) and then washed in TBST. An Olympus confocal laser scanning microscopy system equipped with krypton–argon lasers was used for microscopic observations. The IgG–FITC conjugate was excited at 488 nm. Emission was observed with a 510–560 nm filter. Funding The Human Frontier Science Program Organization [grant to Y.N.M.]. Acknowledgments We thank Peter Westhoff and Susanne von Caemmerer for their gift of seeds of F. bidentis, F. pringlei and F. anomala. Tsuyoshi Furumoto is gratefully acknowledged for providing the cDNA library of F. trinervia and F. pringlei, and seeds of F. trinervia. The antisera against spinach PsbO and AtpE were kindly provided by the late Akira Watanabe and Tohru Hisabori, respectively. 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Proteomics ,  2008, vol.  7 (pg.  1609- 1638) Google Scholar CrossRef Search ADS PubMed  Munekage YN,  Genty B,  Peltier G.  Effect of PGR5 impairment on photosynthesis and growth in Arabidopsis thaliana,  Plant Cell Physiol. ,  2008, vol.  49 (pg.  1688- 1698) Google Scholar CrossRef Search ADS PubMed  Munekage Y,  Hashimoto M,  Miyake C,  Tomizawa K,  Endo T,  Tasaka M, et al.  Cyclic electron flow around photosystem I is essential for photosynthesis,  Nature ,  2004, vol.  429 (pg.  579- 582) Google Scholar CrossRef Search ADS PubMed  Munekage Y,  Hojo M,  Meurer J,  Endo T,  Tasaka M,  Shikanai T.  PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis,  Cell ,  2002, vol.  110 (pg.  361- 371) Google Scholar CrossRef Search ADS PubMed  Pfündel E,  Pfeffer M.  Modification of photosystem I light harvesting of bundle-sheath chloroplasts occurred during the evolution of NADP-malic enzyme C4 photosynthesis,  Plant Physiol. ,  1997, vol.  114 (pg.  145- 152) Google Scholar CrossRef Search ADS PubMed  Rumeau D,  Becuwe-Linka N,  Beyly A,  Louwagie M,  Garin J,  Peltier G.  New subunits NDH-M, -N, and -O, encoded by nuclear genes, are essential for plastid Ndh complex functioning in higher plants,  Plant Cell ,  2005, vol.  17 (pg.  219- 232) Google Scholar CrossRef Search ADS PubMed  Sage RF.  Sage RF,  Monson RK.  Why C4 photosynthesis?,  C4 Plant Biology ,  1999 San Diego Academic Press(pg.  3- 14) Sage RF.  The evolution of C4 photosynthesis,  New Phytol. ,  2004, vol.  161 (pg.  341- 370) Google Scholar CrossRef Search ADS   Takabayashi A,  Kishine M,  Asada K,  Endo T,  Sato F.  Differential use of two cyclic electron flows around photosystem I for driving CO2-concentration mechanism in C4 photosynthesis,  Proc. Natl Acad. Sci. USA ,  2005, vol.  102 (pg.  16898- 16903) Google Scholar CrossRef Search ADS   Abbreviations Abbreviations FITC fluorescein isothiocyanate LHCII light-harvestsing complex II NADP-ME NADP-malic enzyme NDH NAD(P)H dehydrogenase PGR5 PROTON GRADIENT REGULATION 5 RACE rapid amplification of cDNA ends TBST Tris-buffered saline with Tween. © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

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Oxford University Press
Copyright
© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
ISSN
0032-0781
eISSN
1471-9053
DOI
10.1093/pcp/pcq030
pmid
20212018
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Abstract

Abstract Cyclic electron transport around PSI has been proposed to supply the additional ATP required for C4 photosynthesis. To investigate the nature of cyclic electron pathways involved in C4 photosynthesis, we analyzed tissue-specific expression of PGR5 (PROTON GRADIENT REGULATION 5), which is involved in the antimycin A-sensitive pathway, and NDH-H, a subunit of the plastidial NAD(P)H dehydrogenase complex, in four Flaveria species comprising NADP-malic enzyme (ME)-type C4, C3–C4 intermediate and C3 species. PGR5 was highly expressed in the C4 species and enriched in bundle sheath chloroplasts together with NDH-H, suggesting that electron transport of both PGR5-dependent and NDH-dependent cyclic pathways is promoted to drive C4 photosynthesis. C4 photosynthesis requires the coordinated functioning of two cell types, namely mesophyll and bundle sheath cells, with individual functions (Hatch 1987, Sage 1999). Although C4 photosynthesis suppress photorespiration by a CO2-concentrating mechanism, it increases the energetic cost of CO2 assimilation in comparison with C3 photosynthesis. Consequently, two extra ATP molecules are required for each CO2 molecule fixed to drive C4 photosynthesis (Kanai and Edwards 1999). The extra ATP needed for C4 photosynthesis was suggested to be produced by PSI cyclic electron transport activity, which contributes to generation of ΔpH across the thylakoid membrane (Kanai and Edwards 1999). Increased PSI cyclic electron transport compared with C3 plants has been reported in a number of C4 plants, including Sorghum bicolor and Zea mays (Herbert et al. 1990, Asada et al. 1993). Two cyclic pathways around PSI have been identified in C3 plants; the first pathway involves a plastidial NDH [NAD(P)H dehydrogenase] complex that is able to reduce plastoquinones from stromal NAD(P)H donors (Horvath et al. 2000) and the second pathway is an antimycin A-sensitive pathway involving PROTON GRADIENT REGULATION 5 (PGR5), which is localized in the chloroplast and considered to be a factor for major cyclic electron transport activity in C3 plants (Munekage et al. 2002, Munekage et al. 2004, Munekage et al. 2008). The contribution of NDH-dependent electron transport to C4 photosynthesis was suggested by its expression profile correlating with predicted ATP requirement in different cell types (Kubicki et al. 1996, Takabayashi et al. 2005). The contribution of PGR5-related electron transport to C4 photosynthesis is unclear. However, Ivanov et al. (2007) reported that the oxidation level of P700 in bundle sheath strands isolated from Z. mays was dependent on antimycin A. With the aim of investigating the nature of cyclic electron pathways involved in C4 photosynthesis, we analyzed expression of PGR5 and NDH-H, a subunit of the NDH complex, in the dicot genus Flaveria (Asteraceae), which contains closely related C3, C3–C4 intermediate and NADP-malic enzyme (ME)-type C4 species and is a widely used model system for studying the C4 photosynthesis evolutionary process (Sage 2004). The relative abundances of PGR5 and NDH-H were analyzed by immunoblotting in a C3-type species, F. pringlei, a C3–C4 intermediate species, F. anomala, and two NADP-ME C4-type species, F. trinervia and F. bidentis (Fig. 1). The abundance of major electron transport complexes was analyzed using specific antibodies against PsbO for PSII, the Rieske protein for the cytochrome b6f complex, and PsaC for PSI (Fig. 1). The C3F. pringlei and the C3–C4 intermediate F. anomala showed a similar relative abundance of PGR5, NDH-H and subunits of the major electron transport chain. In contrast, relative amounts of PGR5 and NDH-H in both C4 species were four and eight times higher than in C3–C4 intermediate and C3 species, respectively. While PsbO was less abundant in C4 species than in C3 and C3–C4 intermediate species, PsaC was slightly more abundant in C4 species. The abundance of the Rieske protein was similar among all the species compared. Fig. 1 View largeDownload slide Immunoblot analysis of PGR5, NDH-H, PsbO, Rieske protein and PsaC in C4F. trinervia, C4F. bidentis, C3–C4F. anomala and C3F. pringlei. Total membrane proteins were extracted from leaves of each Flaveria species. Lanes were loaded with 20 μg of proteins for detection of PGR5 and NDH-H, 5 μg of protein for detection of PsbO, 10 μg of proteins for detection of Rieske protein and PsaC, and a dilution series of F. bidentis as indicated. Fig. 1 View largeDownload slide Immunoblot analysis of PGR5, NDH-H, PsbO, Rieske protein and PsaC in C4F. trinervia, C4F. bidentis, C3–C4F. anomala and C3F. pringlei. Total membrane proteins were extracted from leaves of each Flaveria species. Lanes were loaded with 20 μg of proteins for detection of PGR5 and NDH-H, 5 μg of protein for detection of PsbO, 10 μg of proteins for detection of Rieske protein and PsaC, and a dilution series of F. bidentis as indicated. To compare the amino acid sequence of PGR5 between C3 and C4 species, the full-length PGR5 gene was cloned from cDNA libraries of F. trinervia, F. bidentis and F. pringlei. Alignment of the deduced protein sequences using the ClustalW algorithm (Supplementary Fig. S1) shows that the sequence is highly conserved among C3 and C4Flaveria. The program TARGETP (www.cbs.dtu.dk/services/TargetP) predicted that the N-terminal sequence of these PGR5 homologs contains a chloroplast-targeted transit peptide. The C-terminal sequence used to raise the PGR5 antibody (from Ala102 to Leu121) was 100% identical among the Flaveria species, indicating that the variation in immunodetection signal intensity reflects actual differences in PGR5 protein levels. To investigate the localization of PGR5 and the NDH complex in C3 and C4Flaveria, in situ immunolabeling was performed using both anti-PGR5 and anti-NDH-H antibodies (Fig. 2). Transverse sections of the leaf lamina of each Flaveria species were stained with toluidine blue. Numerous chloroplasts were colored blue in the mesophyll and bundle sheath cells (Fig. 2A–C). Transverse sections, prepared from the same leaf samples, were labeled with either the pre-immune serum or the immune serum and subsequently labeled with secondary antibodies conjugated to fluorescein isothiocyanate (FITC). Overlaid images of the FITC fluorescence in green and autofluorescence in red were visualized by confocal microscopy. The background labeling with pre-immune serum was very low in all cases (Fig. 2G–H, M, N) compared with the control section (labeling without primary antibody, Fig. 2D–F). There was very little immunolabeling for PGR5 in the C3F. pringlei (Fig. 2J). Although FITC fluorescence was observed in the vascular bundle, which did not contain chloroplasts (Fig. 2J), similar FITC fluorescence patterns were observed in leaf transverse sections of the Arabidopsis pgr5 mutant lacking PGR5 protein (data not shown), indicating that the FITC fluorescence observed in the vascular bundle was not caused by the PGR5 protein. In F. bidentis, more intense immunolabeling for PGR5 was observed in bundle sheath cells compared with mesophyll cells (Fig. 2K). The strong FITC fluorescence superimposed onto chloroplasts resulted in yellow fluorescence and indicated specific immunolabeling for PGR5. A similar result was obtained for F. trinervia (Fig. 2L). These results showed that PGR5 was enriched in bundle sheath chloroplasts of C4Flaveria. Immunolabeling for NDH-H showed exclusive localization of NDH-H in bundle sheath chloroplasts of the C4F. bidentis but only very faint staining of NDH-H in mesophyll cells of the C4F. bidentis and C3F. pringlei (Fig. 2O, P). Fig. 2 View largeDownload slide In situ immunolocalization of PGR5 and NDH-H in leaf tissue of C3F. pringlei (A, D, G, J, M, O), C4F. bidentis (B, E, H, K, N, P) and C4F. trinervia (C, F, I, L). Transverse sections of the lamina for anatomical observation were stained with toluidine blue (A–C). Localization of PGR5 and NDH-H was visualized by the green fluorescence of the FITC-labeled antibody. Leaf sections were stained with primary anti-PGR5 serum (J–L), anti-NDH-H serum (O, P) or without primary antibody (D–F). Pre-immunization sera for PGR5 (G–I) and for NDH-H (M, N) were used to analyze background labeling. Leaf sections were subsequently stained with secondary antibody (anti-rabbit-IgG–FITC conjugate). Overlaid images of green FITC fluorescence and red auto fluorescence were visualized by confocal microscopy. Scale bars = 100 μm. Fig. 2 View largeDownload slide In situ immunolocalization of PGR5 and NDH-H in leaf tissue of C3F. pringlei (A, D, G, J, M, O), C4F. bidentis (B, E, H, K, N, P) and C4F. trinervia (C, F, I, L). Transverse sections of the lamina for anatomical observation were stained with toluidine blue (A–C). Localization of PGR5 and NDH-H was visualized by the green fluorescence of the FITC-labeled antibody. Leaf sections were stained with primary anti-PGR5 serum (J–L), anti-NDH-H serum (O, P) or without primary antibody (D–F). Pre-immunization sera for PGR5 (G–I) and for NDH-H (M, N) were used to analyze background labeling. Leaf sections were subsequently stained with secondary antibody (anti-rabbit-IgG–FITC conjugate). Overlaid images of green FITC fluorescence and red auto fluorescence were visualized by confocal microscopy. Scale bars = 100 μm. In NADP-ME-type C4 photosynthesis, 3.3 ATP/2.1 NADPH molecules are estimated to be required per CO2 molecule fixed in mesophyll cells, which is a similar ratio to that in C3 photosynthesis, whereas only 2.3 ATP per CO2 fixed is considered to be required in bundle sheath cells since NADPH is supplied by decarboxylation of C4 acid (Kanai and Edward 1999). Increased expression of PGR5 and NDH-H in bundle sheath cells of NADP-ME-type C4Flaveria suggested promotion of both PGR5-dependent and NDH-dependent cyclic activities to fulfill the ATP requirement of C4 photosynthesis. In a previous study, expression profiles of NDH-H were well correlated with the predicted ATP requirement in C4 cell types, in contrast to PGR5. This was observed in both NAD-ME-type C4 plants and NADP-type C4 plants (Takabayashi et al. 2005). From these results, it was suggested that the NDH complex mainly energizes C4 photosynthesis. However, if the expression of PGR5 was normalized to the cytochrome f level, whose expression is comparable between mesophyll and bundle sheath cells (Kubicki et al., 1996, Majeran et al., 2008), the PGR5 expression could be correlated with the predicted ATP requirement in C4 cell types in those plants, with the exception of the case of NAD-ME-type Portulaca oleracea (Takabayashi et al. 2005). Here, we showed increased expression of PGR5 from the C3, over the C3–C4 intermediates to the C4 species of the genus Flaveria (Fig. 1B) and used an immunolabeling technique which showed enrichment of PGR5 protein in bundle sheath cells (Fig. 2). This result suggests that a PGR5-dependent pathway contributes to ATP production, which drives C4 photosynthesis. However, the abundances of PGR5, NDH-H and major electron transport complexes were similar between the C3–C4 intermediate F. anomala and C3F. pringlei. The proportion of PSII/PSI is similar between C3–C4 intermediate species and C3 species of Flaveria (Pfündel and Pfeffer 1997). Together, these findings suggest that the composition of the electron transport chain has remained unchanged during the evolution of C3 to C3–C4 intermediate species in Flaveria. In C4Flaveria, two morphologically distinct chloroplast types were observed, as in S. bicolor and Z. mays (Laetsch 1971, Hofer et al. 1992). While mesophyll chloroplasts contain numerous grana thylakoid membranes, bundle sheath chloroplasts contain grana-free thylakoid membranes (Laetsch 1971). Enrichment of PGR5 on bundle sheath chloroplasts suggests that PGR5 is probably localized on non-appressed thylakoid membranes. To test this hypothesis, thylakoid membranes isolated from C3F. pringlei or C4F. bidentis leaves were fractionated into grana and stroma lamellae by digitonin treatment (Cuello and Quiles 2004). SDS–PAGE revealed protein patterns typical for grana and stroma lamellae in C3F. pringlei; light- harvesting complex II (LHCII) was enriched in the grana thylakoid fraction, and α and β subunits of ATP synthase were enriched in the stroma lamellae fraction (Fig. 3A). A similar protein pattern was observed in the C4 species, with the exception of LHCIIs, which were also detected in the stroma lamellae fraction (Fig. 3A). Immunoblotting against Lhcb1 and the ε subunit of ATP synthase (AtpE) showed that Lhcb1 and AtpE were mainly localized in the grana thylakoid fraction and stroma lamellae fractions, respectively, in both F. pringlei and F. bidentis, but a small amount of Lhcb1 was also detected in stroma lamellae in F. bidentis (Fig. 3B). A higher amount of Lhcb1 was detected in the stroma lamellae fraction of F. bidentis than that of F. pringlei, which is probably due to localization of LHCIIs in agranal thylakoid membranes of bundle sheath chloroplasts in C4Flaveria (Hofer et al. 1992). Immunoblotting against PGR5 showed that PGR5 was enriched in the stroma lamellae fraction in both F. pringlei and F. bidentis (Fig. 3B). Identical results were obtained with tobacco (data not shown). From these results, we concluded that PGR5 is localized in stroma lamellae. Fig. 3 View largeDownload slide Distribution of PGR5 in grana and stroma thylakoids in C3F. pringlei and C4F. bidentis. (A) Coomassie blue staining of proteins extracted in the total thylakoids fraction (T), grana fraction (G) and stroma lamellae fraction (S). (B) Immunoblotting of PGR5, Lhcb1 and AtpE. The lanes were loaded with 10 μg of protein for Coomassie blue staining and immunoblotting of PGR5, and 0.5 μg of protein for immunoblotting of Lhcb1 and AtpE. Fig. 3 View largeDownload slide Distribution of PGR5 in grana and stroma thylakoids in C3F. pringlei and C4F. bidentis. (A) Coomassie blue staining of proteins extracted in the total thylakoids fraction (T), grana fraction (G) and stroma lamellae fraction (S). (B) Immunoblotting of PGR5, Lhcb1 and AtpE. The lanes were loaded with 10 μg of protein for Coomassie blue staining and immunoblotting of PGR5, and 0.5 μg of protein for immunoblotting of Lhcb1 and AtpE. Localization of PGR5 in stroma lamellae indicates that PGR5-related cyclic electron transport takes place in non-appressed thylakoid membranes. It also explains why NADP-ME-type C4 species developed agranal chloroplasts in bundle sheath cells to promote PSI cyclic electron transport. Interestingly, while expression of the PSI subunit was slightly higher in C4 species than in C3 species, expression of PGR5 was four times higher in C4 species than in C3 species (Fig. 1). We propose that the increased expression of PGR5 and the NDH complex directly promotes cyclic electron transport activity to drive ATP production for C4 photosynthesis in C4Flaveria species. Materials and Methods Plants of F. trinervia, F. bidentis, F. anomala and F. pringlei were grown in pots on soil for 6–8 weeks in a growth chamber (200–300 μmol photons m−2 s−1, 16 h light/8 h dark, 25–28°C). A cDNA library was prepared from leaves of F. bidentis. Total RNA was extracted from leaves using the RNeasy Maxi Kit (Qiagen, Courteboeuf, France) and poly(A) mRNA was isolated with the PolyATtract® mRNA Isolation System IV (Promega, Madison, WI, USA). cDNA synthesis and rapid amplification of cDNA ends (RACE) (3′ and 5′) PCR was performed using the Marathon™ cDNA Amplification Kit (Clontec, Ozyme, France) according to the manufacturer’s instructions. Primers for RACE PCR are listed in Supplementary Table S1. Leaf samples were blended in liquid N2 and the powder was suspended in 50 mM Tris–HCl buffer (pH 8.0) containing 50 mM β-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride. The membrane fraction was sedimented by centrifugation at 27,000 × g for 15 min and the pellet was resuspended in 50 mM Tris–HCl buffer (pH 8.0) containing 1% SDS. Stromal and granal thylakoid membrane separation was performed essentially as described by Rumeau et al. (2005). Leaves were homogenized at high speed using a Polytron homogenizer to grind mesophyll and bundle sheath cells. Isolated thylakoids were resuspended in 100 mM Tricine/NaOH buffer (pH 7.8) containing 10 mM NaCl and 10 mM MgCl2 to a chlorophyll concentration of 1.5 mg ml−1 and incubated for 3 min with digitonin (Sigma-Aldrich) added to give a final concentration of 8.25 mg ml−1. The samples were centrifuged at 10,000 × g for 30 min to separate grana membranes. After centrifugation at 40,000 × g for 30 min to remove contaminating grana vesicles, the supernatant (stroma lamellae) was sedimented at 150,000 × g for 1 h. Denaturing SDS–PAGE was performed using 15% (w/v) polyacrylamide gels. Proteins were electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with the following antibodies. The anti-NDH-H antibody raised from recombinant proteins and the anti-PGR5 antibody raised from a synthetic peptide antigen are described in Horvath et al. (2000) and Munekage et al. (2002), respectively. The anti-PsaC antibody and anti-Lhcb1 antibody were purchased from Agrisera (Vannas, Sweden). Antibodies against Rieske were raised from recombinant proteins (Sanda et al. unpublished data). Immunocomplexes were detected using the ECL Plus Western Blot Detection Reagent (GE Healthcare). Small leaf tissue fragments were first fixed in 3.7% formaldehyde, 5% acetic acid and 50% ethanol, dehydrated with ethanol, impregnated with xylene and then embedded in paraffin. Embedded tissue was sliced into 8 μm sections and mounted onto glass slides, deparaffinized and stained with toluidine blue for anatomical observation. For immunolabeling, deparaffinized sections were incubated in blocking solution [Tris-buffered saline with Tween (TBST) with 3% dry milk] then incubated with or without primary antibody (1 : 300 diluted in blocking solution). After washing in TBST, the sections were incubated with the secondary antibody (anti-rabbit-IgG–FITC conjugate, Sigma-Aldrich, 1 : 500) and then washed in TBST. An Olympus confocal laser scanning microscopy system equipped with krypton–argon lasers was used for microscopic observations. The IgG–FITC conjugate was excited at 488 nm. Emission was observed with a 510–560 nm filter. Funding The Human Frontier Science Program Organization [grant to Y.N.M.]. Acknowledgments We thank Peter Westhoff and Susanne von Caemmerer for their gift of seeds of F. bidentis, F. pringlei and F. anomala. Tsuyoshi Furumoto is gratefully acknowledged for providing the cDNA library of F. trinervia and F. pringlei, and seeds of F. trinervia. The antisera against spinach PsbO and AtpE were kindly provided by the late Akira Watanabe and Tohru Hisabori, respectively. 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Natl Acad. Sci. USA ,  2005, vol.  102 (pg.  16898- 16903) Google Scholar CrossRef Search ADS   Abbreviations Abbreviations FITC fluorescein isothiocyanate LHCII light-harvestsing complex II NADP-ME NADP-malic enzyme NDH NAD(P)H dehydrogenase PGR5 PROTON GRADIENT REGULATION 5 RACE rapid amplification of cDNA ends TBST Tris-buffered saline with Tween. © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Journal

Plant and Cell PhysiologyOxford University Press

Published: Apr 8, 2010

Keywords: C 4 photosynthesis Cyclic electron transport Flaveria

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